Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin
Introduction
Influenza A viruses are responsible for recurrent seasonal epidemics as well as periodic global pandemics associated with acute respiratory disease and death [1]. The epidemiological success of these viruses is due in large part to a segmented genome which allows them to evolve by exchange, or reassortment, of gene segments between different influenza A strains. When reassortment involves an exchange of one of the major surface proteins, it is known as antigenic shift and the virus that emerges usually presents the human immune system with such a novel antigenic experience that it results in high morbidity and/or mortality [2]. The most clinically profound antigenic shifts involve the key protective antigen, hemagglutinin (HA). Thus, novel influenza A subtypes arising from antigenic shift, particularly those involving HA, can lead to severe pandemics due to the absence of pre-existing protective immunity in individuals and the population as a whole [1], [3].
Highly virulent influenza A variants often emerge in birds or swine and then cross species barriers to infect humans [3], [4], [5], [6], [7], [8], [9]. Currently, the highly pathogenic avian influenza (HPAI) viruses circulating in Asia are regarded as having the potential for crossing the species barrier and causing a pandemic [10], [11], [12]. In 1997, the first human case involving HPAI of the H5N1 subtype was reported in Southeast Asia. Subsequently, over 360 human cases of HPAI of the H5 subtype, with a case-fatality rate over 60%, have been reported [13], [14], [15]. Although poultry-to-human transmission remains the predominant means of transmission, public health risk may increase significantly if the human-to-human barrier is overcome by a reassortant virus.
Vaccines are central to pandemic preparedness. However, two barriers to the development and production of vaccines that address global pandemic influenza needs have emerged. First, the immunological potency of the HA associated with pandemic strains is significantly less than that observed for the seasonal human influenza strains [16], [17]. Second, existing influenza manufacturing processes that use either eggs or eukaryotic cell culture are inefficient and require large centralized, committed manufacturing facilities that are not amenable to the rapid production of stockpiles sufficient for national or global requirements [18], [19], [20]. We have developed an approach that addresses these barriers by linking the Toll-like receptor (TLR) 5 agonist, flagellin, to a self-sufficient protective subunit of HA.
Toll-like receptors (TLRs) are expressed on various cell types, including professional antigen presenting cells, where they act as primary sensors of microbial infection and then activate signaling cascades that lead to the induction of immune responses [21], [22]. It is well established that physical linkage of TLR ligands and vaccine antigens enhances the immunopotency of the linked antigen [23]. In previous studies, we have demonstrated that physical linkage of vaccine antigens to flagellin generates a significantly more potent vaccine than simple mixing of antigen and flagellin [24], [25], [26].
More recently, we have used this approach to generate novel influenza vaccines that fuse the globular head of HA with flagellin [27]. The globular head domain spans the majority of the neutralizing epitopes in HA and, in the context of the flagellin fusion protein, stably refolds to faithfully form these conformationally sensitive epitopes. These fusion proteins are immunologically potent vaccines that can be efficiently manufactured at scales to meet global needs using standard E. coli fermentation systems. We have now extended this approach to the development and evaluation of several prototypic pandemic vaccines based on the A/Vietnam/1203/2004 H5 strain (VN04). We find that replacement of domain D3 of flagellin with the globular head domain of the Vietnam HA leads to the generation of a highly effective vaccine in the mouse lethal challenge model.
Section snippets
Tissue and egg culture
The Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cell lines (American Type Culture Collection, Manassas, VA) were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum and antibiotics. SPAFAS Specific Pathogen Free premium eggs were supplied by Charles River Laboratories (Wilmington, MA).
Viruses
Animal infections and viral assays were performed with influenza A/Vietnam/1203/04 (Influenza Laboratory, U.S. Centers for Disease Control and
Rationale for vaccine designs
The HA globular head domain contains the cell surface receptor binding site and the majority of the neutralizing antibody epitopes [23], [33]. We have recently demonstrated that when the H1 HA globular head is fused to the C-terminus of flagellin, the recombinant influenza vaccines elicited HA specific antibody responses and protected mice from the lethal challenge [27]. Here, we have developed a similar approach to pandemic vaccines by generating a similar subunit vaccine which encompasses the
Discussion
We have evaluated several H5 subtype pandemic vaccines based on the influenza A/Vietnam/1203/2004 (VN04) virus. The vaccines all comprise the TLR5 ligand, flagellin, fused to the globular head domain of the VN04 HA and are efficiently manufactured using standard prokaryotic fermentation systems. We find that the R3 vaccine, in which domain D3 of flagellin is replaced with the HA globular head, is most effective at eliciting protective HAI titers and protecting mice from disease and death.
Based
Acknowledgments
We are grateful to Drs. Phil Wyde, Alan Shaw, Bob Becker and David Taylor for providing scientific input and technical training and to Jenna Linde for excellent data entry through the studies and for assistance in preparing the manuscript figures. We also thank Nathaniel Linde for technical assistance and Dr. Diana Noah and her team for performing the HAI assay at the BSL3 facility in Southern Research Institute. SP was supported by NIH K08 grant no. AI059491. Funding was provided by a grant
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Cited by (0)
- 1
These two authors contribute equally to this work.
- 2
Current address: Sanofi Pasteur, Swiftwater, PA, United States.